Internalization is required for proper Wingless signaling in Drosophila melanogaster

Abstract

The Wnt–Wingless (Wg) pathway regulates development through precisely controlled signaling. In this study, we show that intracellular trafficking regulates Wg signaling levels. In Drosophila melanogaster cells stimulated with Wg media, dynamin or Rab5 knockdown causes reduced Super8XTOPflash activity, suggesting that internalization and endosomal transport facilitate Wg signaling. In the wing, impaired dynamin function reduces Wg transcription. However, when Wg production is unaffected, extracellular Wg levels are increased. Despite this, target gene expression is reduced, indicating that internalization is also required for efficient Wg signaling in vivo. When endosomal transport is impaired, Wg signaling is similarly reduced. Conversely, the expression of Wg targets is enhanced by increased transport to endosomes or decreased hepatocyte growth factor–regulated tyrosine kinase substrate– mediated transport from endosomes. This increased signaling correlates with greater colocalized Wg, Arrow, and Dishevelled on endosomes. As these data indicate that endosomal transport promotes Wg signaling, our findings suggest that the regulation of endocytosis is a novel mechanism through which Wg signaling levels are determined.

Frizzled and LRP5/6 Receptors for Wnt/ -Catenin Signaling

“…(2) Because GSK3 levels do not overtly decrease during Wnt signaling, it requires a mechanism for GSK3 subsequently to escape MVBs, which normally shuttles the cargo to lysosomes for degradation. (3) It suggests that genes involved in MVB formation are required for Wnt signaling, for which supporting evidence exists in Xenopus but not in Drosophila (Piddini et al 2005;Rives et al 2006;Seto and Bellen 2006;Taelman et al 2010). (4) It predicts that most GSK3 substrates (in fact, 20% of cellular proteins) in addition to b-catenin are under Wnt regulation (Taelman et al 2010), thus departing from the prevailing view that roles of GSK3 in Wnt and other signaling (such as insulin) pathways are insulated from one another.…”

“…Thus, some cautions are warranted, particularly when perturbations of general endocytic pathways are performed and thus broad cellular events beyond Wnt signaling are affected (Gagliardi et al 2008). Indeed, manipulating endocytic molecules in Drosophila has also yielded contradictory results regarding Wg signaling (Piddini et al 2005;Rives et al 2006;Seto and Bellen 2006). Finally, some of the cell biological studies of LRP6 and Wnt signaling have relied on overexpression of LRP6 and other components.…”

Endocytosis of Wingless via a dynamin-independent pathway is necessary for signaling in Drosophila wing discs

“…Perturbation of CD endocytosis by using temperature-sensitive mutants or DN mutants of shibire (fly homolog of Dynamin) was found to affect Wg signaling in wing discs (11,13). To characterize the DFz2-independent internalization route of Wg, we examined the extent of endocytosis of DFz2 and Wg in wing discs of the temperature-sensitive mutant shibire ts1 (shi ts1 ) larvae after incubating them at restrictive temperatures (31-32°C) for a short interval of time (15 min).…”

“…However, rescue of patterning is observed even upon replacement of the endogenous Wg with a transmembrane-tethered Wg, thus raising questions on the importance of a secreted Wg gradient (15). Regardless, inhibition of endocytosis in the recipient cells, by using the dominant-negative (DN) or the temperature-sensitive form of shibire, demonstrates the importance of dynamin-dependent endocytosis in Wg-mediated signaling (11,13). Interestingly, when examined carefully, Wg is observed in endosomes even in null clones of its signaling receptors Frizzled (Fz) and Arrow (14,(16)(17), suggesting that other receptors or pathways may be important for its internalization.…”

“…Perturbation of any of these processes leading to Wg secretion results in both accumulation of Wg within the producing cells and reduction of Wg signaling in the receiving zone of the wing disc (9,10). Endocytosis in the signal-receiving cells may either be important in shaping the distribution of secreted Wg across the wing disc (11) or in a cell-autonomous fashion affect signaling by promoting the interaction of Wg and DFz2 within an endosome (12,13). Endocytosis also mediates Arrow-directed degradation necessary for the observed Wg distribution and signaling (14).…”